Process for the preparation and purification of thiol-containing maytansinoids

ABSTRACT

The present invention provides a process for the preparation and purification of thiol-containing maytansinoids comprising the steps of: (1) reductive hydrolysis of a maytansinoid C-3 ester with a reducing agent selected from the group consisting lithium trimethoxyaluminum hydride (LiAl(OMe) 3 H), lithium triethoxyaluminum hydride (LiAl(OEt) 3 H), lithium tripropoxyaluminum hydride (LiAl(OPr) 3 H), sodium trimethoxyaluminum hydride (NaAl(OMe) 3 H), sodium triethoxyaluminum hydride (NaAl(OEt) 3 H) and sodium tripropoxyaluminum hydride (NaAl(OPr) 3 H) to yield a maytansinol; (2) purifying the maytansinol to remove side products when present; (3) esterifying the purified maytansinol with a carboxylic acid to yield a mixture of an L- and a D-aminoacyl ester of maytansinol; (4) separating the L-aminoacyl ester of maytansinol from the reaction mixture in (3); (5) reducing the L-aminoacyl ester of maytansinol to yield a thiol-containing maytansinoid; and (6) purifying the thiol-containing maytansinoid.

FIELD OF THE INVENTION

The present invention relates to a process for preparing and purifyingcytotoxic agents. More specifically, the invention relates a process forpreparing and purifying cytotoxic agents comprising thiol-containingmaytansinoids. These cytotoxic agents can be used as therapeutic agentsby linking them to a cell binding agent, through the thiol group, andthen delivering them to a specific cell population in a targetedfashion.

BACKGROUND OF THE INVENTION

In recent years, a myriad of reports have appeared on the attemptedspecific targeting of tumor cells with monoclonal antibody-drugconjugates (R. V. J. Chari., 31 Adv. Drug Deliv. Res., 89-104 (1998); G.A. Pietersz and K. Krauer, 2 J. Drug Targeting 183-215 (1994); Sela etal., in Immunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al., inTargeted Drugs 1-22 (E. Goldberg, ed. 1983); Diener et al., in Antibodymediated delivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et al.,in Antibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988);Bumol et al., in Antibody mediated delivery system 55-79 (J. Rodwell,ed. 1988). Cytotoxic drugs such as methotrexate, daunorubicin,doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, andchlorambucil have been conjugated to a variety of murine monoclonalantibodies. In some cases, the drug molecules were linked to theantibody molecules through an intermediary carrier molecule such asserum albumin (Garnett et al., 46 Cancer Res. 2407-2412 (1986); Ohkawaet al., 23 Cancer Immumol. Immunother. 81-86 (1986); Endo et al., 47Cancer Res. 1076-1080 (1980)), dextran (Hurwitz et al., 2 Appl. Biochem.25-35 (1980); Manabi et al., 34 Biochem. Pharmacol. 289-291 (1985);Dillman et al., 46 Cancer Res. 4886-4891 (1986); Shoval et al., 85 Proc.Natl. Acad. Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada etal., 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al., 27 J. Med.Chem. 1602-1607 (1984); Tsukada et al., 52 Br. J. Cancer 111-116(1985)).

A wide array of linker technologies have been employed for thepreparation of such immunoconjugates and both cleavable andnon-cleavable linkers have been investigated. In most cases, the fullcytotoxic potential of the drugs could only be observed, however, if thedrug molecules could be released from the conjugates in unmodified format the target site.

One of the cleavable linkers that has been employed for the preparationof antibody-drug conjugates is an acid-labile linker based oncis-aconitic acid that takes advantage of the acidic environment ofdifferent intracellular compartments such as the endosomes encounteredduring receptor mediated endocytosis and the lysosomes. Shen and Ryserintroduced this method for the preparation of conjugates of daunorubicinwith macromolecular carriers (102 Biochem. Biophys. Res. Commun.1048-1054 (1981)). Yang and Reisfeld used the same technique toconjugate daunorubicin to an anti-melanoma antibody (80 J. Natl. Canc.Inst. 1154-1159 (1988)). Dillman et al. also used an acid-labile linkerin a similar fashion to prepare conjugates of daunorubicin with ananti-T cell antibody (48 Cancer Res. 6097-6102 (1988)). Trail et al.linked doxorubicin to antibodies via an acid-labile hydrazone bond (52Cancer Res. 5693-5700 (1992)).

An alternative approach, explored by Trouet et al., involved linkingdaunorubicin to an antibody via a peptide spacer arm (79 Proc. Natl.Acad. Sci. 626-629 (1982)). This was done under the premise that freedrug could be released from such a conjugate by the action of lysosomalpeptidases.

In vitro cytotoxicity tests, however, have revealed that antibody-drugconjugates rarely achieved the same cytotoxic potency as the freeunconjugated drugs. This suggested that mechanisms by which drugmolecules are released from the antibodies are very inefficient. In thearea of immunotoxins, conjugates formed via disulfide bridges betweenmonoclonal antibodies and catalytically active protein toxins were shownto be more cytotoxic than conjugates containing other linkers. See,Lambert et al., 260 J. Biol. Chem. 12035-12041 (1985); Lambert et al.,in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al., 48 CancerRes. 2610-2617 (1988). This was attributed to the high intracellularconcentration of glutathione contributing to the efficient cleavage ofthe disulfide bond between an antibody molecule and a toxin. Despitethis, there are only a few reported examples of the use of disulfidebridges for the preparation of conjugates between drugs andmacromolecules. Shen et al. described the conversion of methotrexateinto a mercaptoethylamide derivative followed by conjugation withpoly-D-lysine via a disulfide bond (260 J. Biol. Chem. 10905-10908(1985)). A recent report described the preparation of a conjugate of thetrisulfide-containing toxic drug calicheamicin with an antibody (L. M.Hinman et al., 53 Cancer Res. 3336-3342 (1993); E. L. Sievers et al., 93Blood 3678-3684 (1999).

One reason for the lack of disulfide linked antibody-drug conjugates isthe unavailability of cytotoxic drugs possessing a sulfur atomcontaining moiety that can be readily used to link the drug to anantibody via a disulfide bridge. Furthermore, chemical modification ofexisting drugs is difficult without diminishing their cytotoxicpotential.

Another major drawback with existing antibody-drug conjugates is theirinability to deliver a sufficient concentration of drug to the targetsite because of the limited number of targeted antigens and therelatively moderate cytotoxicity of cancerostatic drugs likemethotrexate, daunorubicin and vincristine. For example, an antibodyconjugate of doxorubicin was evaluated in human clinical trials, andfound to be ineffective (Tolcher et al., 17 J. Clinical Oncol. 478-484(1999)). In order to achieve significant cytotoxicity, linkage of alarge number of drug molecules either directly to the antibody orthrough a polymeric carrier molecule becomes necessary. However, suchheavily modified antibodies often display impaired binding to the targetantigen and fast in vivo clearance from the blood stream.

Maytansinoids are highly cytotoxic drugs. Maytansine was first isolatedby Kupchan et al. from the east African shrub Maytenus serrata and shownto be 100 to 1000 fold more cytotoxic than conventional cancerchemotherapeutic agents like methotrexate, daunorubicin, and vincristine(U.S. Pat. No. 3,896,111). Subsequently it was discovered that somemicrobes also produce maytansinoids, such as maytansinol and C-3 estersof maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 esters ofmaytansinol and analogues of maytansinol have also been reported(Kupchan et al., 21 J. Med. Chem. 31-37 (1978); Higashide et al., 270Nature 721-722 (1977); Kawai et al., 32 Chem. Pharm. Bull. 3441-3451(1984)). Examples of analogues of maytansinol from which C-3 esters havebeen prepared include maytansinol with modifications on the aromaticring (e.g. dechloro) or at the C-9, C-14 (e.g. hydroxylated methylgroup), C-15, C-18, C-20 and C-4,5.

The naturally occurring and synthetic C-3 esters can be classified intotwo groups:

(a) C-3 esters with simple carboxylic acids (U.S. Pat. Nos. 4,248,870;4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and4,331,598), and

(b) C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat. Nos.4,137,230; 4,260,608; 5,208,020; 5,416,064; and 12 Chem. Pharm. Bull.3441 (1984)).

Esters of group (b) were found to be much more cytotoxic than esters ofgroup (a).

Maytansine is a mitotic inhibitor. Treatment of L1210 cells in vivo withmaytansine has been reported to result in 67% of the cells accumulatingin mitosis. Untreated control cells were reported to demonstrate amitotic index ranging from between 3.2 to 5.8% (Sieber et al., 43Comparative Leukemia Research 1975, Bibl. Haemat. 495-500 (1976)).Experiments with sea urchin eggs and clam eggs have suggested thatmaytansine inhibits mitosis by interfering with the formation ofmicrotubules through the inhibition of the polymerization of themicrotubule protein, tubulin (Remillard et al., 189 Science 1002-1005(1975)).

In vitro P388, L1210, and LY5178 murine leukemic cell suspensions havebeen found to be inhibited by maytansine at doses of 10⁻³ to 10⁻¹microgram/mL, with the P388 line being the most sensitive. Maytansinehas also been shown to be an active inhibitor of in vitro growth ofhuman nasopharyngeal carcinoma cells. The human acute lymphoblasticleukemia line C.E.M. was reported inhibited by concentrations as low as10⁻⁷ microgram/ml (Wolpert-DeFillippes et al., 24 Biochem. Pharmacol.1735-1738 (1975)).

In vivo, maytansine has also been shown to be active. Tumor growth inthe P388 lymphocytic leukemia system was shown to be inhibited over a50- to 100-fold dosage range which suggested a high therapeutic index;also significant inhibitory activity could be demonstrated with theL1210 mouse leukemia system, the human Lewis lung carcinoma system andthe human B-16 melanocarcinoma system (Kupchan, 33 Ped. Proc. 2288-2295(1974)).

Because the maytansinoids are highly cytotoxic, they were expected to beof use in the treatment of many diseases such as cancer. Thisexpectation has yet to be realized. Clinical trials with maytansine werenot favorable due to a number of side effects (Issel et al., 5 Can.Trtmnt. Rev. 199-207 (1978)). Adverse effects to the central nervoussystem and gastrointestinal symptoms were responsible for some patientsrefusing further therapy (Issel at 204), and it appeared that maytansinewas associated with peripheral neuropathy that might be cumulative(Issel at 207).

However, forms of maytansinoids that are highly cytotoxic, yet can stilleffectively be used in the treatment of many disease, have beendescribed (U.S. Pat. Nos. 5,208,020 and 5,416,064; Chari et al., 52Cancer Res. 127-131 (1992); Liu et al., 93 Proc. Natl. Acad. Sci.8618-8623 (1996)).

A further drawback to the therapeutic use of maytansinoids, of interesthere, is that the process for the preparation and purification ofthiol-containing maytansinoids involves several inefficientchromatographic steps that are cumbersome, not easily scalable andresult in only moderate yields.

U.S. Pat. Nos. 5,208,020 and 5,416,064 disclose that a thiol-containingmaytansinoid may be produced by first converting a maytansinoid bearingan ester group into maytansinol, then esterifying the resultingmaytansinol with N-methyl-L-alanine or N-methyl-L-cysteine derivativesto yield disulfide-containing maytansinoids, followed by cleavage of thedisulfide group with dithiothreitol to the thiol-containingmaytansinoids. However, this process involves several inefficient stepsthat are cumbersome and result in moderate yields.

More specifically, maytansinol is first derived from maytansine or otheresters of maytansinol by reduction, such as with lithium aluminumhydride. (Kupchan, S. M. et al., 21 J. Med. Chem. 31-37 (1978); U.S.Pat. No. 4,360,462). It is also possible to isolate maytansinol from themicroorganism Nocardia (see Higashide et al., U.S. Pat. No. 4,151,042).In a specific example, the conversion of Ansamitocin P-3 intomaytansinol by reductive hydrolysis with lithium aluminum hydride intetrahydrofuran at −5° C. was described in U.S. Pat. No. 4,162,940.However, the reaction with lithium aluminum hydride results in theformation of several side products which can be removed only by carefulpreparative thin layer chromatography on silica gel. Thus, this processis not amenable for industrial scale use.

The next step in the process is the conversion of maytansinol todifferent ester derivatives using N-methyl-L-alanine orN-methyl-L-cysteine derivatives, and suitable agents such asdicyclohexylcarbodiimide (DCC) and catalytic amounts of zinc chloride(see U.S. Pat. No. 4,137,230; Kawai et al., 32 Chem. Pharm. Bull.3441-3951 (1984); U.S. Pat. No. 4,260,609). Two diastereomeric productscontaining the D and L-aminoacyl side chains result, as does a smallportion of unreacted maytansinol. While the unreacted maytansinol isreadily separated from its esters by column chromatography on silicagel, the diastereomeric maytansinoid esters are barely separable. In theprocess previously described (Kupchan, S. M., 21 J. Med. Chem. 31-37(1978); U.S. Pat. No. 4,360,462), the desired L-aminoacyl ester isobtained after purification over two silica gel columns followed byfurther purification by preparative thin layer chromatography on silicagel. Thus, this process is also not amenable for industrial scale use.

The last step in the process, the reduction of the disulfide-containingmaytansinoids to the corresponding thiol-containing maytansinoids, isachieved by treatment with dithiotlireitol (DTT), purification by HPLCusing a C-18 column and elution with a linear gradient of 55% to 80%acetonitrile in H₂O. However, this step results in low yields and isalso not amenable for industrial scale use. Thiol-containingmaytansinoids are not very soluble in the ethanol/water solvent mixtureused for the reaction. Furthermore, purification by HPLC on a reversephase C-18 column using acetonitrile/water as the mobile phase resultsin poor recovery and can result in the dimerization of some product.

Accordingly, an improved process for the preparation and purification ofthiol-containing maytansinoids, that reduces the complexity of theprocess, allows scalability and increases the yield, is greatly needed.

SUMMARY OF THE INVENTION

Thus, the object of the present invention is to provide an improvedprocess for the preparation and purification of thiol-containingmaytansinoids that reduces the complexity of the process, allowsscalability and improves the yield.

This and other objectives have been achieved by providing an improvedprocess for the preparation and purification of thiol-containingmaytansinoids.

In one embodiment, the present invention provides a process forpreparing a thiol-containing maytansinoid comprising the steps of:

(1) conducting reductive hydrolysis of a maytansinoid C-3 ester with areducing agent selected from the group consisting of lithiumtrimethoxyaluminum hydride (LiAl(OMe)₃H), lithium triethoxyaluminumhydride (LiAl(OEt)₃H), lithium tripropoxyaluminum hydride (LiAl(OPr)₃H),sodium trimethoxyaluminum hydride (NaAl(OMe)₃H), sodiumtriethoxyaluminum hydride (NaAl(OEt)₃H) and sodium tripropoxyaluminumhydride (NaAl(OPr)₃H), to yield a maytansinol;

(2) purifying the maytansinol to remove side products when present;

(3) esterifying the purified maytansinol with a carboxylic acid to yielda reaction mixture of an L- and a D-aminoacyl ester of maytansinol;

(4) separating the L-aminoacyl ester of maytansinol from the reactionmixture in (3);

(5) reducing the L-aminoacyl ester of maytansinol to yield athiol-containing maytansinoid; and

(6) purifying the thiol-containing maytansinoid.

Preferably, the reducing agent in (1) is lithium trimethoxyaluminumhydride.

Also preferably, the reducing agent in (1) is used in a concentration offrom about 5 to 100 equivalents per mole of the maytansinoid C-3 ester.More preferably, the reducing agent in (1) is used in a concentration offrom about 7.5 to 30 equivalents per mole of the maytansinoid C-3 ester.Most preferably, the reducing agent in (1) is used in a concentrationfrom about 10 to 20 equivalents per mole of the maytansinoid C-3 ester.

Preferably, the reductive hydrolysis in (1) is conducted at atemperature of from about −80° C. to 0° C. More preferably, thereductive hydrolysis in (1) is conducted at a temperature of from about−45° C. to −27.5° C. Most preferably, the reductive hydrolysis in (1) isconducted at a temperature of from about −35° C. to −30° C.

Preferably, the reducing agent in (1) is added over a period of fromabout 5 to 40 minutes. More preferably, the reducing agent in (1) isadded over a period of from about 7 to 20 minutes. Most preferably, thereducing agent in (1) is added over a period of from about 8 to 12minutes.

Preferably, the maytansinol is purified in (2) by chromatography. Morepreferably, the maytansinol is purified in (2) by chromatography wherethe chromatography is silica gel column chromatography, preparativethin-layer chromatography on silica gel or cyano-bonded silica HPLCcolumn chromatography. Most preferably, the maytansinol is purified in(2) by chromatography where the chromatography is silica gel columnchromatography.

Preferably, the purification in (2) is performed at ambient temperature.

Preferably, the maytansinol in (2) is purified to a purity of about 95%.

Preferably, the carboxylic acid in (3) is selected from the groupconsisting of N-methyl-N-methyldithioacetyl-L-alanine,N-methyl-N-(3-methyldithio-propanoyl)-L-alanine,N-methyl-N-(3-methyldithio-butanoyl)-L-alanine,N-methyl-N-(4-methyldithio-butanoyl)-L-alanine,N-methyl-N-(5-methyldithio-pentanoyl)-L-alanine,N-methyl-N-(3-phenyldithio-propanoyl)-L-alanine,N-methyl-N-[3-(4-nitrophenyldithio)-propanoyl]L-alanine,N-acetyl-N-methyl-methyldithiocysteine andN-acetyl-N-methylmethyldithiohomocysteine. More preferably, thecarboxylic acid in (3) isN-methyl-N-(3-methyldithio-propanoyl)-L-alanine.

Preferably, the esterification in (3) is conducted at ambienttemperature.

Preferably, the esterification in (3) further comprises the use ofdicyclohexylcarbodiimide and zinc chloride.

Preferably, the separating in (4) is carried out by passing the reactionmixture over a cyano-bonded silica HPLC column.

Preferably, the separating in (4) is carried out at about 25° C.

Preferably, the reduction in (5) uses dithiothreitol as the reducingagent.

Preferably, the reduction in (5) is carried out in a mixture of ethylacetate-methanol-aqueous buffer which is capable of keeping buffersalts, dithiothreitol, unreduced maytansinoids and reduced maytansinoidsin solution. More preferably, the mixture of ethylacetate-methanol-aqueous buffer is 1:1.5:1, v/v/v, ethylacetate:methanol:aqueous buffer.

Preferably, the concentration of the thiol-containing maytansinoid issuch that the thiol-containing maytansinoid remains soluble in ethylacetate-methanol-aqueous buffer. Preferably, the concentration of thethiol-containing maytansinoid is about 4 g/L.

Preferably, the reduction in (5) is carried out in an oxygen-freeatmosphere.

Preferably, the reduction in (5) is carried out at about 25° C.

Preferably, the purification of the thiol-containing maytansinoid in (6)is by chromatography. More preferably, the chromatography is by acyano-bonded HPLC column. Most preferably, the chromatography is by acyano-bonded HPLC column equilibrated and run in an organic solvent.Preferably, the organic solvent is a mixture of hexanes:2-propanol:ethylacetate, more preferably the solvent is a 78.0:5.5:16.5, v/v/v, mixtureof hexanes:2-propanol:ethyl acetate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reduction of ansamitocins 1 a-e to yield maytansinol(2).

FIG. 2a shows the conversion of the ω-mercapto-carboxylic acids 3 a-e tothe respective methyl-dithio derivatives 4 a-e.

FIG. 2b shows the conversion of the ω-mercapto-carboxylic acid 3 b tothe respective aryl-dithio derivatives 5 a-b.

FIG. 2c shows the conversion of the methyl-dithio derivatives 4 a-e andthe aryl-dithio derivatives 5 a-b to N-methyl-L-alanine derivatives 6a-g containing a disulfide group.

FIG. 3a shows the conversion of N-methylcysteine andN-methylhomocysteine (7 a-b) to the respective disulfide derivatives 8a-b.

FIG. 3b shows the acylation of the disulfide derivatives 8 a-b toN-methyl-L-cysteine derivatives 9 a-b containing a disulfide group.

FIG. 4 shows the esterification of maytansinol (2) with theN-methyl-L-alanine derivatives 6 a-g containing a disulfide group toyield the disulfide-containing maytansinoid stereoisomers 10, from whichthe L-isomers are separated via chromatography to yield thedisulfide-containing maytansinoid L-isomers 10 a-g, which in turn arereduced to yield the thiol-containing maytansinoids 11 a-g.

FIG. 5 shows the esterification of maytansinol (2) with theN-methyl-L-cysteine derivatives 9 a-b containing a disulfide group toyield the disulfide-containing maytansinoid stereoisomers 12, from whichthe L-isomers are separated via chromatography to yield thedisulfide-containing maytansinoid L-isomers 12 a-b, which in turn arereduced to yield the thiol-containing maytansinoids 13 a-b.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the synthesis of thiol-containingmaytansinoid derivatives that retain high cytotoxicity and that can beeffectively linked to cell binding agents. The art reveals that theexisting methods for producing thiol-containing maytansinoids arecomplex, non-scalable and produce low product yield. The presentinvention overcomes these problems by disclosing a novel process forproducing thiol-containing maytansinoids that reduces the complexity ofthe process, allows scalability and improves product yield.

Thus, the invention provides a novel process for the production ofthiol-containing maytansinoids, useful agents for the elimination ofdiseased or abnormal cells that are to be killed or lysed, such as tumorcells (particularly solid tumor cells), virus infected cells,microorganism infected cells, parasite infected cells, autoimmune cells(cells that produce autoantibodies), activated cells (those involved ingraft rejection or graft vs. host disease), or any other type ofdiseased or abnormal cells, while exhibiting a minimum of side effects.

These thiol-containing maytansinoids can be chemically linked to a cellbinding agent while keeping a high cytotoxicity either in bound form orin released form or in both states. High cytotoxicity is defined asexhibiting a toxicity having an IC₅₀—the inhibiting concentration of atoxic substance that leaves a surviving fraction of 0.5—of about 10⁻⁸ Mor less when measured in vitro with KB cells upon a 24 hour exposuretime to the drug.

The effectiveness of the compounds of the invention as therapeuticagents depends on the careful selection of an appropriate cell bindingagent. Cell binding agents may be of any kind presently known, or thatbecome known and include peptides and non-peptides. Generally, these canbe antibodies (especially monoclonal antibodies), lymphokines, hormones,growth factors, nutrient-transport molecules (such as transferrin), orany other cell binding molecule or substance.

Suitable Maytansinoids

There have been a number of disclosures that teach the production ofmaytansinoids and maytansinoid derivatives that may be used as acytotoxic agent. Examples of suitable maytansinoids include maytansinoland maytansinol analogues. Examples of suitable maytansinol analoguesinclude those having a modified aromatic ring and those havingmodifications at other positions.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring, and the process for their production, include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by lithiumaluminum hydride reduction of ansamitocin P-2);

(2) C-20-hydroxy (or C-20-demethyl) ±C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (—OCOR), ±dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H₂S or P₂S₅);

(2) C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora);

(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

In order to link the maytansinoid to the cell binding agent, a linkinggroup is used.

Suitable linking groups are well known in the art and include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred aredisulfide groups and thioether groups.

According to the present invention the linking group is part of achemical moiety that is covalently bound to the maytansinoid throughdisclosed methods. In a preferred embodiment, the chemical moiety iscovalently bound to the maytansinoid via an ester linkage.

Many positions on maytansinoids are expected to be useful as the linkageposition, depending upon the type of link. For example, for forming anester linkage, the C-3 position having a hydroxyl group, the C-14position modified with hydroxymethyl, the C-15 position modified withhydroxy and the C-20 position having a hydroxy group are all expected tobe useful. However the C-3 position is preferred and the C-3 position ofmaytansinol is especially preferred.

Also preferred is an N-methyl-L-alanine-containing C-3 ester ofmaytansinol and an N-methyl-L-cysteine-containing C-3 ester ofmaytansinol or their analogues.

The present invention recites a process for the preparation andpurification of a thiol-containing maytansinoid that positions thelinking group at C-3 of maytansinol.

Step 1

Synthesis of Maytansinol (2) from an Ester-Bearing Maytansinoid

The first step in the preparation and purification of thiol-containingmaytansinoids involves the reductive hydrolysis of a maytansinoid C-3ester into maytansinol (2).

A) A maytansinoid C-3 ester is dissolved in an anhydrous solvent, thenplaced under an inert gas atmosphere and cooled.

Preferably, the maytansinoid C-3 ester is maytansine or ansamitocin P-3(1 c). Any ansamitocin (1 a-e), such as ansamitocin P-4, P-3, P-2 andP-1 (described in Asai et al., 35 Tetrahedron 1079-1085 (1979); U.S.Pat. No. 4,450,234; Hatano et al., 48 Agric. Biol. Chem. 1721-1729(1984)) can also be used, more preferably ansamitocin P-3 (1 c).Maytansine can obtained as described by Kupchan et al., 42 J. Org. Chem.2349-2357 (1977). The microbiological preparation of ansamitocin P-3 (1c) is described in U.S. Pat. No. 4,450,234 and in Hatano et al., 48Agric. Biol. Chem. 1721-1729 (1984).

Preferably, the anhydrous solvent is tetrahydrofuran (THF),2-methoxyethyl ether, dioxane or di-ethyl ether, although other solventscan be used. More preferably, the anhydrous solvent is tetrahydrofuran.Preferably, 20 to 30 mL of the anhydrous solvent is used per gram ofester, more preferably 25 mL/g. Preferably, the inert gas is argon,nitrogen or helium, although other gases can be used. More preferably,the inert gas is argon. Preferably, the solution is maintained atbetween about 0 to −80° C., more preferably between about −30 to −50°C., most preferably between about −35 to −45° C., in a dry ice-acetonebath.

B) A reducing agent is also cooled, and then transferred into thechilled solution of the maytansinoid C-3 ester. The reaction ismaintained under an inert gas atmosphere, at a low temperature, andstirred.

Preferably, the reducing agent is lithium trimethoxyaluminum hydride(LiAl(OMe)₃H), lithium triethoxyaluminum hydride (LiAl(OEt)₃H), lithiumtripropoxyaluminum hydride (LiAl(OPr)₃H), sodium trimethoxyaluminumhydride (NaAl(OMe)₃H), sodium triethoxyaluminum hydride (NaAl(OEt)₃H) orsodium tripropoxyaluminum hydride (NaAl(OPr)₃H). More preferably, thereducing agent is lithium trimethoxyaluminum hydride (LiAl(OMe)₃H).Preferably, the reducing agent is cooled to about −30 to −40° C. in adry ice-acetone bath. Preferably, the cooled reducing agent istransferred into the chilled solution of the maytansinoid C-3 ester viaa canula. Preferably, the inert gas is argon, nitrogen or helium,although other gases can be used. More preferably, the inert gas isargon.

Preferably, the reducing agent is used in a concentration of from about5 to 100 equivalents per mole of the maytansinoid C-3 ester, morepreferably from about 7.5 to 30 equivalents per mole, most preferablyfrom about 10 to 20 equivalents per mole. One of ordinary skill in theart will understand that use of reducing agent in amounts greater thanabout 100 equivalents per mole of the maytansinoid C-3 ester can resultin undesired side products. Preferably, the reducing agent is added tothe chilled solution of maytansinoid C-3 ester over a time periodranging from about 5 to 40 minutes, more preferably from about 7 to 20minutes, most preferably from about 8 to 12 minutes. Preferably, thereaction is maintained under an inert gas atmosphere at a temperaturerange of from about −25° C. to −80° C., more preferably from about −27°C. to −45° C., most preferably from about −30° C. to −35° C. Preferably,the reaction is stirred between about 30 min and three hours, morepreferably for about three hours.

One of ordinary skill in the art will understand that the amount ofreducing agent used, the temperature maintained during the reaction, thelength of the time period over which the reducing agent is added and thereaction time are each dependent on the other. For example, the lowerthe amount of the reducing agent, the longer the reaction time.Similarly, the lower the temperature, the larger the excess of reducingagent required and the longer the time required for completion of thereaction. Moreover, the slower the rate the reducing agent is added, thelonger reaction time required for completion of the reaction.

C) The reaction is quenched, extracted, dried and filtered. The solventis evaporated under reduced pressure to yield crude maytansinol.

Preferably, the reaction is quenched by the addition of saturated sodiumchloride solution, water or ammonium chloride solution, more preferablyby the addition of saturated sodium chloride solution. Preferably, thereaction is quenched using between about 20 to 40 mL of the solution pergram of maytansinoid ester used. Preferably, the reaction is extractedwith ethyl acetate, dichloromethane, toluene, chloroform or ether, morepreferably with ethyl acetate. Preferably, the reaction is extracted atthe rate of between about 4×80 to 4×200 mL/g maytansinoid ester used.Preferably, the combined ethyl acetate extracts are dried over sodiumsulfate or magnesium sulfate, more preferably sodium sulfate, andfiltered.

D) The crude maytansinol (2) may be purified, if required, bychromatography.

In a preferred embodiment, the crude maytansinol (2) may be purified bydissolving it in a minimum volume of ethyl acetate, dichloro methane,ether, chloroform or toluene, more preferably ethyl acetate, andpurified via silica gel column chromatography using dichloromethane,chloroform, ethyl acetate or toluene, more preferably dichloromethane.Preferably, the maytansinol (2) is eluted with a step gradient startingwith dichloromethane or ethyl acetate, or a mixture ofdichloromethane:ethyl acetate:alcohol, chloroform:ethyl acetate ortoluene:ethyl acetate, preferably 77:23:0, v/v/v, dichloromethane:ethylacetate:alcohol. The concentration of alcohol is slowly increased from 0to about 20%, preferably from 0 to about 10%. Fractions containing thedesired product are pooled and evaporated under reduced pressure toyield pure maytansinol (2) as a white solid. The purification may beperformed at ambient temperature. Preferably, the purification isperformed at a temperature of about 20° C. and 25° C. Preferably, thealcohol is methanol, ethanol, n-propanol, iso-propanol, n-, iso-, sec-,or tert-butanol, more preferably methanol.

In another preferred embodiment, the crude maytansinol (2) may bepurified using a cyano-bonded silica HPLC column that is run in normalphase, using organic solvents in the mobile phase. The crude maytansinol(2) is dissolved in ethyl acetate, ethyl acetate:isopropanol:hexane(mobile phase) or in ethyl acetate-isopropanol, preferably ethylacetate, injected onto the column, and the appropriate peak iscollected. Preferably, the cyano-bonded HPLC column that is used for theseparation is one having cyanopropyl and cyano-di-isopropyl groupsstably bonded to the silica backbone. Such columns are available underthe trade names Diazem™, Zorbax™, Monochrom™, and Kromasil™, to name afew. Preferably, the organic solvent used in the mobile phase ishexanes:2-propanol:dichloromethane:ethyl acetate. Preferably, theconcentration of each constituent of the organic solvent is in the range65-75:2-4:15-20:5-10, v/v/v/v, respectively. More preferably, theconcentration of each element of the organic solvent is 72:3:18:7,v/v/v/v.

Preferably, maytansinol (2) purified by this process is at least 90%pure, more preferably at least 95% pure. One of ordinary skill in theart will understand that if maytansinol (2) of less than 90% purity isused in the following steps, additional purification steps may berequired.

One of ordinary skill in the art will understand that the reductionreaction may yield small amounts of undesired side products, in additionto the maytansinol (2). Thus, purification may be required to remove thecontaminants. However, if the side products are not generated, such aswhere the precise times, temperatures, and amounts are established, thenthe purification step is not required.

Step 2

Synthesis of Esters of Maytansinol (2) Having a Linking Group

The maytansinol (2) from Step 1 is next esterified withdisulfide-containing N-methyl-L-alanine derivatives 6 a-g orN-methyl-L-cysteine derivatives 9 a-b, in the presence ofdicyclohexylcarbodiimide (DCC) and zinc chloride, to yield L- andD-aminoacyl esters 10 a-g and 12 a-b of maytansinol, containing adisulfide-linking group. The stereoisomers are then separated, and theL-aminoacyl ester is collected. Because the esterification reactionresults in some epimerization at the chiral center of the amino acidderivatives 6 a-g and 9 a-b, one of ordinary skill in the art willunderstand that the racemic versions of 6 a-g and 9 a-b (i.e. a D,L-mixture) can be used in Step 2, in place of the stereoisomers.

A) In one embodiment, the N-methyl-L-alanine derivatives 6 a-gcontaining a disulfide group are produced by the conversion ofω-mercaptocarboxylic acids 3 a-e of varying chain lengths into theirrespective methyl-dithio, e.g. 4 a-e (where n=1-4, including branchedand cyclic aliphatics), or aryl-dithio, e.g. 5 a-b, derivatives byreacting them with methyl methanethiolsulfonate or aryldisulfides, suchas diphenyldisulfide, and ring substituted diphenyldisulfides andheterocyclic disulfides, such as 2,2′-dithiopyridine. These carboxylicacids are then reacted with N-methyl-L-alanine to form the desiredN-methyl-L-alanine derivatives containing a disulfide group 6 a-g thatwill be condensed with maytansinol (2) to form maytansinoids containingdisulfide-linking groups 10 a-g.

N-methyl-L-alanine may be prepared as described in the literature (see,Fu, S. J. & Birnbaum, S. M., 75 J. Amer. Chem. Soc. 918 (1953)), or isobtainable commercially (Sigma Chemical Company).

Preferably, the N-methyl-L-alanine derivatives containing a disulfidegroup are N-methyl-N-methyldithioacetyl-L-alanine,N-methyl-N-(3-methyldithio-propanoyl)-L-alanine,N-methyl-N-(4-methyldithio-butanoyl)-L-alanine,N-methyl-N-(3-methyldithio-butanoyl)-L-alanine,N-methyl-N-(5-methyldithio-pentanoyl)-L-alanine,N-methyl-N-(3-phenyldithio-propanoyl)-L-alanine andN-methyl-N-[3-(4-Nitrophenyldithio)-propanoyl]L-alanine.

B) In another embodiment, N-methyl-L-cysteine (7 a) orN-methyl-L-homocysteine (7 b) can be converted to the respectivedisulfide derivatives 8 a-b (n=1 and 2, respectively), which are thenacylated to yield the desired N-methyl-L-cysteine orN-methyl-L-homocysteine derivatives containing a disulfide group 9 a-b(n=1 and 2, respectively). These disulfide-containing derivatives 9 a-bwill be condensed with maytansinol (2) to form maytansinoids containingdisulfide-linking groups 12 a-b.

N-methyl-L-cysteine can be prepared as described in Undheim and Eidem,23 Acta Chem. Scand. 3129-3133 (1970).

Preferably, the N-methyl-L-cysteine derivatives containing a disulfidegroup are N-acetyl-N-methyl-methyldithiocysteine (9 a) andN-acetyl-N-methyl-methyldithiohomocysteine (9 b).

C) Maytansinol (2) is esterified with a N-methyl-L-alanine orN-methyl-L-cysteine derivative containing a disulfide group (6 a-g and 9a-b) by first preparing a solution of the disulfide-containingderivative. This solution is stirred under an inert gas atmosphere, andtreated sequentially with solutions of (a) DCC or EDC([3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride),preferably DCC, (b) ZnCl₂ and (c) maytansinol. The mixture is stirreduntil completion of the reaction. After the reaction is complete, it isfiltered and the filtrate evaporated under reduced pressure yielding amixture of the L- and D-aminoacyl esters 10 and 12 of maytansinol.

As noted above, because the esterification reaction results in someepimerization at the chiral center of the amino acid derivatives 6 a-gand 9 a-b, one of ordinary skill in the art will understand that theracemic versions of 6 a-g and 9 a-b (i.e. a D, L-mixture) can be used inplace of the stereoisomers.

Preferably, the N-methyl-L-alanine or N-methyl-L-cysteine derivativecontaining a disulfide group isN-methyl-N-(methyldithio-propanoyl)-L-alanine (6 b) andN-acetyl-N-methyl-methyldithiocysteine (9 a), respectively. Preferably,the solution of the disulfide-containing derivative comprises drymethylene chloride, tetrahydrofuran, or ether, preferably dry methylenechloride, using between about 10 to 50 mL/gram maytansinol, preferably20 mL/gram. Preferably, the inert gas is argon, nitrogen or helium,although other gases can be used. More preferably, the inert gas isargon.

Preferably the DCC or EDC is in methylene chloride, tetrahydrofuran orether, preferably methylene chloride, using about 10 to 15 mL per gramDCC or EDC, preferably 12 mL/g. Preferably, the DCC or EDC solution isadded at the rate of about 5 to 7 moles/mole of maytansinol. Preferably,the ZnCl₂ is about 1 M, and it is used at between about 1.2 to 1.5moles/mole of maytansinol, preferably 1.25 moles, in ether or methylenechloride, preferably ether. Preferably, maytansinol is in methylenechloride, tetrahydrofuran or ether, more preferably methylene chloride,using about 10 to 120 mL solvent per gram of maytansinol, preferably 100mL/g. The reaction mixture is stirred at between 4 to 30° C., preferablyroom temperature, for between 1 to 24 hours, preferably three hours. Oneof ordinary skill in the art will understand that the length of time thereaction requires stirring is dependent on the temperature, with lowertemperatures resulting in longer reaction time.

D) Separation of the L- and D-aminoacyl maytansinol esters 10 a-g and 12a-b is accomplished via a cyano-bonded silica HPLC column that is run innormal phase, using organic solvents in the mobile phase. A solution ofthe L- and D-aminoacyl maytansinol ester mixture, dissolved in ethylacetate or in hexanes:2-propanol:ethyl acetate, is injected on to thecolumn. The mobile phase can be adjusted to obtain separation inretention times of the two isomers that is in excess of 10 minutes.

Alternatively, the separation can be conducted using silica gelchromatography, but the process is not easily converted to an industrialscale. Separation can also be accomplished through other, lessdesirable, means such as HPLC using chiral columns or silica columns.

Preferably, the cyano-bonded HPLC columns that are used for theseparation are those that have cyanopropyl and cyano-di-isopropyl groupsstably bonded to the silica backbone. Such columns are available underthe trade names Diazem™, Zorbax™, Monochrom™, and Kromasil™, amongothers. Preferably, the organic solvent used in the mobile phase ishexanes:2-propanol:ethyl acetate. Preferably, the yield of theseparation is greater than about 90% and product is at least about 95%pure, more preferably, optically pure. Under the aforementionedconditions, the desired L-isomer has a retention time of about 36 to 46min, preferably 42 min, while the D-isomer elutes at about 50 to 60minutes, preferably 56 min. Preferably, the esterification is carriedout at about 25° C.

Step 3

Synthesis of Thiol-Containing Maytansinoid 11 a-g and 13 a-b

To obtain thiol-containing maytansinoids 11 a-g and 13 a-b, theL-aminoacyl maytansinol esters obtained from Step 2 are dissolved in asolution comprising a solvent and an alcohol. This mixture is stirredunder an inert gas atmosphere, and treated with a solution ofdithiothreitol or dithioerythritol, in buffer, and containingethylenediaminetetraacetic acid (EDTA). The progress of the reaction maybe monitored by HPLC and is generally complete in 3 h, although it mayvary between 1 and 24 h. The completed reaction is treated with abuffered solution containing EDTA, and then extracted. The organiclayers are combined, washed and dried. Evaporation of the solvent leavesa residue of crude thiol-containing maytansinoid 11 a-g and 13 a-b,which may be purified using a preparative cyano-bonded silica HPLCcolumn. Fractions containing the product may be evaporated to yield purethiol-containing maytansinoid as a white solid.

Preferably, the solvent the L-aminoacyl maytansinol esters obtained fromStep 2 are dissolved in is ethyl acetate, dichloromethane or ether, morepreferably ethyl acetate, using from about 60 to 80 mL solvent per grammaytansinoid, preferably 72 mL/g. Preferably, the alcohol theL-aminoacyl maytansinol esters obtained from Step 2 are dissolved in ismethanol or ethanol, more preferably methanol, using from about 90 to120 mL alcohol per gram of maytansinoid, preferably 108 mL/g.Preferably, the reaction between L-aminoacyl maytansinol esters anddithiothreitol is in a mixture of ethyl acetate:methanol:aqueous bufferwhich is capable of keeping the buffer salts, dithiothreitol andmaytansinoids (reduced and non-reduced forms) in solution. Morepreferably, the reaction between L-aminoacyl maytansinol esters anddithiothreitol is in a 1:5:1 mixture of ethyl acetate:methanol:aqueousbuffer.

Preferably, the concentration of L-aminoacyl maytansinol esters used inStep 3 is less than about 4 g/L such that the L-aminoacyl maytansinolesters remain solubilized.

Preferably, the reduction reaction is carried out in an oxygen-freeatmosphere. Preferably, the inert gas is argon, nitrogen or helium,although other gases can be used. More preferably, the inert gas isargon.

Preferably, the reduction reaction is carried out at between 4 to 30°C., more preferably at room temperature. One of ordinary skill in theart will understand that the reaction can be carried out at lowertemperatures, however, the time required for completion of the reactionis increased.

Preferably, the solution containing the L-aminoacyl maytansinol estersis treated with dithiothreitol, dithioerythritol or a phosphine reagentsuch as tris(2-carboxyethyl)phosphine (TCEP), more preferablydithiothreitol, using about 2 to 3 moles reducing agent per molemaytansinoid, preferably 2.5 moles/mole, in potassium phosphate buffer,sodium phosphate buffer or triethanolamine buffer, preferably potassiumphosphate buffer, the buffer at a concentration between about 20 to 100mM, preferably 50 mM, using 60 to 80 mL of the buffered DTT per grammaytansinoid, preferably 72 mL/g, and containing from about 1 to 10 mM,preferably 2 mM, ethylenediaminetetraacetic acid (EDTA).

Preferably, the completed reaction is treated with a 0.2 M solution ofpotassium phosphate buffer, sodium phosphate buffer or triethanolaminebuffer, more preferably potassium phosphate buffer, using from about 120to 160 mL buffer per gram maytansinoid, preferably 144 mL/g, andcontaining from about 1 to 10 mM, preferably 2 mM, EDTA. Preferably, theextraction is with ethyl acetate, dichloro methane, or ether, morepreferably ethyl acetate, using 200 to 500 mL per gram maytansinoid,preferably 300 mL/g, repeated three times. Preferably, the combinedorganic layers are washed with a saturated sodium chloride solution,water or a saturated ammonium chloride solution, more preferably asaturated sodium chloride solution, using 40 to 100 mL per grammaytansinoid, preferably 50 mL/g, and then dried over sodium sulfate ormagnesium sulfate, preferably sodium sulfate.

Preferably, the cyano-bonded HPLC columns that are used for theseparation are those that have cyanopropyl and cyano-di-isopropyl groupsstably bonded to the silica backbone. Such columns are available underthe trade names Diazem™, Zorbax™, Monochrom™, and Kromasil™, to name afew. More preferably, a Diazem preparative CN column (250 mm×50 mm, 10micron particle size) is used. Preferably, the organic solvent used inthe mobile phase is hexanes:2-propanol:ethyl acetate. More preferably,the organic solvent used in the mobile phase is a 78.0:5.5:16.5 (v/v/v)mixture of hexanes:2-propanol:ethyl acetate. Preferably, the flow rateis 150 mL/min. Preferably, the yield of the separation is greater than75%, and product is at least about 90% pure, more preferably, at leastabout 95% pure. Preferably, the desired thiol-containing maytansinoids11 a-d and 13 a-b have a retention time of 16 min, within a range fromabout 14 to 18 min.

Specific examples of N-methyl-L-alanine-containing maytansinoidderivatives taught by the present invention are represented by formulas(I)-(IV):

wherein: Z represents H or SR, wherein R represents a methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic group; l represents an integer of 1 to 10; and mayrepresents a maytansinoid.

wherein: R₁ and R₂, which may be the same or different, represents H,CH₃ or CH₂CH₃; Z represents H or SR, wherein R represents methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl, orheterocyclic; m represents 0, 1, 2 or 3; and may represents amaytansinoid.

wherein: Z represents H or SR, wherein R represents methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl, orheterocyclic; p represents an integer of 3 to 8; and may represents amaytansinoid.

wherein: Z represents H or SR, wherein R represents methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic; q represents an integer from 1 to 10; Y represents Cl orH; and X represents H or CH₃.

Specific examples of N-methyl-L-cysteine-containing maytansinoidderivatives useful in the present invention are represented by formulas(V) and (VI):

wherein: Z represents H or SR, wherein R represents methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl, orheterocyclic; o represents 1 or 2; q represents 0 or an integer of 1 to10; and may represents a maytansinoid.

wherein: Z represents H or SR, wherein R represents methyl, linearalkyl, branched alkyl, cyclic alkyl, simple or substituted aryl orheterocyclic; o represents 1 or 2; q represents 0 or an integer of 1 to10; Y represents Cl or H; and X represents H or CH₃.

Examples of linear alkyls include methyl, ethyl, propyl, butyl, pentyland hexyl.

Examples of branched alkyls include isopropyl, isobutyl, sec.-butyl,tert.-butyl, isopentyl and 1-ethyl-propyl.

Examples of cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyland cyclohexyl.

Examples of simple aryls include phenyl and naphthyl.

Examples of substituted aryls include aryls such as those describedabove substituted with alkyl groups, with halogens, such as Cl, Br, F,nitro groups, amino groups, sulfonic acid groups, carboxylic acidgroups, hydroxy groups and alkoxy groups.

Examples of heterocyclics are compounds wherein the heteroatoms areselected from O, N and S, and include pyrrollyl, pyridyl, furyl andthiophene.

DEFINITIONS

As used herein, the terms “room temperature” and “ambient temperature”mean environmental temperature or uncontrolled temperature. The term“optical purity” means that the purity exceeds 98%. Where ranges arespecified herein, e.g. temperature, time, concentration etc., the rangeincludes all specific values within the range, as well as subrangesfalling within the broad range. Where values are stated to be “about” aspecific value, or range of values, it is to be understood thatstatistically insignificant variations in those values are included aswell.

EXAMPLES

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percents, ratios, parts, etc. areby weight.

Example 1

Synthesis of Thiol-Containing Maytansinoid Derivatives

Melting points were determined on a electrothermal melting pointapparatus. Proton magnetic resonance (¹H NMR) spectra were obtained on aVarian™ EM360 spectrometer at 60 MHz or on a Bruker™ AM300 machine at300 MHz. Chemical shifts are reported in δ values relative to aninternal tetramethylsilane (TMS) standard. UV spectra were recorded on aPerkin Elmer™ λ4A spectrophotometer. Optical rotations were determinedusing a Perkin Elmer™ model 241 polarimeter. A Rainin™ HP, HewlettPackard™, or Hitachi™ instrument equipped with wavelength or diode arrayUV detector and a Waters™ Radialpak C-18 column or Diazem™ cyano orChromasil™ cyano column was used for HPLC analyses and purification.Elemental analyses were performed by Atlantic Microlabs, Atlanta, GA.

2-Mercaptoacetic acid (3 a), 3-mercaptopropanoic acid (3 b) and4-mercaptobutanoic acid (3 c) are commercially available.

5-Mercaptopentanoic acid (3 d). 5-Mercaptopentanoic acid (3 d) wasprepared by a modification of a literature method (Khim et al, 37 J.Org. Chem. 2714-2720 (1972)). To a stirred solution of 5-bromopentanoicacid (1.80 g, 0.01 mol) in absolute ethanol (25 mL) was added thiourea(0.761 g, 0.01 mol) and the reaction mixture was refluxed for 6 hours. Asolution of 50% aqueous NaOH (20 mL) was then added and the mixturerefluxed for an additional two hours. The mixture was then diluted withwater (100 mL), acidified (aqueous HCl), and extracted with ethylacetate (4×50 mL). The combined organic layers were dried over sodiumsulfate and evaporated under reduced pressure. The residue waschromatographed over silica gel eluting with methylene chloride/ethylacetate to give 0.885 g (66%) of a colorless liquid. ¹H NMR (CDCl₃) δ1.3(1H, t), 1.6 (4H, m), 2.4 (4H, m), 11.5 (1H, s).

3-Mercaptobutanoic acid (3 e). A solution of the commercially availableβ-butyrolactone (3.0 g, 35.0 mmol) in tetrahydrofuran (20 mL) was addedto di-isopropylethyl amine (10.0 g, 77.5 mmol). The reaction mixture wasstirred under an argon atmosphere, and treated with thioacetic acid (3.0g, 39.0 mmol). The reaction mixture was stirred at room temperature for6 h. The solvent was then evaporated and the product purified by columnchromatography over silica gel, eluting with ethyl:hexanes (3:1, v/v)containing 3% acetic acid. The thioacetate derivative of 3 e wasobtained as a colorless oil (2.0 g, 35% yield). ¹H NMR (CDCL₃): δ1.5(3H, d), 2.3 (3H, s), 2.8 (2H, d), 4.0 (1H, m) and 11.3 (1H, s). Thethioacetate was converted into the thiol 3 e by base hydrolysis. Asolution of the thioacetate (1.5 g, 9.3 mmol) in methanol (6 mL) wastreated with a solution of 3.3 M sodium hydroxide in water (10 mL). Thereaction was stirred under an argon atmosphere. The progress of thereaction was monitored by TLC, and was judged complete in 3 h. Thereaction mixture was acidified by addition of 1 M hydrochloric acid (40mL), and extracted with ethyl acetate (70 mL). The organic extract wasdried over sodium sulfate, filtered, and evaporated under reducedpressure to give 3-mercaptobutanoic acid (3 e), which was used withoutfurther purification for the synthesis of 4 e.

Methyldithio-acetic acid (4 a). To a stirred solution of mercaptoaceticacid (3 a) (3.0 g, 0.0326 mmol) in water (100 mL), cooled in an icebath, was added methyl methanethiosulfonate (4.52 g, 0.036 mol) inabsolute ethanol (40 mL). The reaction mixture was allowed to warm toroom temperature and stirred overnight. The reaction mixture was thendiluted with saturated, aqueous NaCl (300 mL), and extracted withdiethyl ether (3×100 mL). The combined organic layer was washed withsaturated sodium chloride (100 mL), and dried over Na₂SO₄, and filtered.The filtrate was evaporated under reduced pressure, and the residuedistilled under vacuum to yield 4 a as a colorless oil (2.90 g, 64%yield) bp_(1mm)=100° C. ¹H NMR: δ2.4 (3H, s), 3.5 (3H, s) and 10.2 (1H,s).

3-Methyldithio-propanoic acid (4 b). To a stirred solution of3-mercaptopropanoic acid (3 b) (5.00 g. 0.047 mol) in water (150 mL),cooled in an ice bath, was added methyl methanethiosulfonate (6.54 g.0.052 mol) in absolute ethanol (75 mL). The reaction mixture was stirredovernight at room temperature. The mixture was then diluted withsaturated, aqueous NaCl (400 mL) and extracted with ether (3×150 mL).The combined ether extracts were washed with saturated NaCl, dried overNa₂SO₄ and concentrated. The residue was distilled to afford a colorlessliquid (6.47 g, 90% yield): bp_(1.0)105° C. ¹H NMR (CDCl₃) δ2.3 (3H, s),2.8 (4H, m), 11.2 (1H, s).

4-Methyldithio-butanoic acid (4 c). To a stirred solution ofbis-(3-carboxypropyl)-disulfide (1.00 g, 4.20 mmol) in methanol (20 mL)was added a solution of dithiothreitol (0.647 g, 4.20 mmol) in H₂O (20mL). A solution of 10M NaOH (0.842 mL, 8.42 mmol) was then added and themixture was allowed to stir at room temperature overnight to effectcomplete reduction. Methyl methanethiolsulfonate (1.17 g, 9.24 mmol) wasadded and the reaction mixture allowed to stir for another three hours.The mixture was then diluted with saturated, aqueous NaCl (150 mL),acidified (aqueous HCl), and extracted with ethyl ether (3×100 mL). Thecombined organic layers were washed with saturated NaCl, dried (Na₂SO₄),concentrated and the concentrate was chromatographed on silica geleluting with methylene chloride/ethyl acetate to give 0.867 g (56%) of aclear liquid. ¹H NMR (CDCl₃) δ2.1 (2H, m), 2.4 (3H, s), 2.4 (2H, m), 2.7(2H, m), 11.1 (1H, s).

5-Methyldithio-pentanoic acid (4 d). To a stirred solution of5-mercaptopentanoic acid (3 d) (0.500 g, 3.73 mmol) in water (20 mL) wasadded a solution of methyl methanethiosulfonate (0.517 g, 4.10 mmol) inabsolute ethanol (5 mL) and the mixture was stirred at room temperaturefor 3 hours. The mixture was then diluted with aqueous, saturated NaCl(100 mL) and extracted with ethyl ether (3×100 mL). The combined organiclayers were washed with saturated NaCl, dried (Na₂SO₄), evaporated underreduced pressure and the concentrate was chromatographed over silicaeluting with methylene chloride/ethyl acetate to yield 0.602 g (90%)white crystals: mp 42-44° C. ¹H NMR (CDCl₃) δ1.7 (4H, m), 2.4 (3H, s),2.4 (2H, m), 2.7 (2H, m), 11.1 (1H, s).

3-Methyldithio-butanoic acid (4 e). A solution of 3 e (1.1 g, 9.3 mmol)in ethyl acetate (10 mL) was treated sequentially with di-isopropylethylamine (3.4 g, 27 mmol) and a solution of methyl methanethiosulfonate(1.5 g, 12.0 mmol) in absolute ethanol (8 mL). The reaction mixture wasstirred at room temperature under argon for 3 h. The solvent was thenevaporated, and the residue treated with 3 M hydrochloric acid (15 mL)and extracted with ethyl acetate (40 mL). The organic layer wasseparated, dried over sodium sulfate, filtered and evaporated. Theresidue was purified by column chromatography over silica gel, elutingwith ethyl acetate:hexanes (3:1, v/v) containing 3% acetic acid. Theproduct 4 e was obtained as a colorless oil (0.61 g, 40% yield). ¹H NMR(CDCl₃): δ1.4 (3H, d), 2.3 (3H, s), 2.8 (2H, d), 3.9 (1H, m) and 11.5(1H, s).

3-Phenyldithio-propanoic acid (5 a). To a stirred solution of diphenyldisulfide (3.0 g, 13.8 mmol) in a mixture of ether (10 mL) and methanol(20 mL), under a nitrogen atmosphere, at room temperature was added asolution of 3-mercaptopropanoic acid (3 b) (0.49 g, 4.6 mmol) in ether(5 mL), followed by a solution of 10M NaOH (0.46 mL, 4.6 mmol). Thereaction mixture was stirred at room temperature for 20 hours, thenstripped of the solvents under reduced pressure. The product waspurified by column chromatography on silica gel eluting with ethylacetate/hexane. The product was obtained as a white solid (0.56 g,56.6%), mp 57-59° C. ¹H NMR (CDCl₃, TMS) δ2.6-3.0 (4H, m), 7.1-7.6 (5H,m), 10.6 (1H, s).

3-(4-Nitrophenyldithio)-propanoic acid (5 b). To a stirred solution ofbis-(4-nitrophenyl)-disulfide (3.00 g, 9.73 mmol) dissolved in a mixtureof THF (200 mL) and methanol (50 mL) was added 3-mercaptopropanoic acid(3 b) (0.688 g, 6.49 mmol). One drop of a solution of 10 N NaOH was thenadded to the mixture and the reaction stirred for one hour. The reactionmixture was then diluted with saturated NaCl (100 mL) and extracted withethyl acetate (3×75 mL). The combined organic layers were dried oversodium sulfate, evaporated under reduced pressure and the product waschromatographed over silica gel eluting with methylene chloride/ethylacetate to yield 0.885 g (53%) of a light yellow solid; mp 98-100° C. ¹HNMR (CDCOCD₃) δ2.8 (2H, m), 3.1 (2H, m), 7.8 (2H, d), 8.2 (2H, d).

N-methyl-N-methyldithioacetoyl-L-alanine (6 a). To a stirred solution of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.99 g,15.6 mmol) and triethylamine (1.58 g, 15.6 mmol) in dry CH₂Cl₂ (40 mL)at 0° C. was added a solution of methyldithio-acetic acid (Singh et al.,104 Anal. Biochem. 51-58 (1980) (4 a) (1.66 g, 12.0 mmol) in dry CH₂Cl₂(20 mL). A solution of 4-dimethylaminopyridine (0.073 g, 0.60 mmol) indry CH₂Cl₂ (2 mL) was added and the mixture stirred for 45 min. at 0° C.A mixture of N-methyl-L-alanine (0.619 g, 6.00 mmol) and triethylamine(0.607 g, 6.00 mmol) in dry DMF (30 mL) was then added and the mixturestirred at 0° C. for two hours. The reaction mixture was diluted withwater (100 mL), stirred for another thirty minutes, then acidified(aqueous HCl) and extracted with ethyl acetate (4×75 mL). The combinedorganic layers were washed several times with water, dried over Na₂SO₄,and evaporated under reduced pressure. The residue was chromatographedover silica gel eluting with methylene chloride ethyl acetate to yield0.25 g (19%) of a pale yellow oil. ¹H-NMR (CDCl₃) δ1.4 (3H, d), 2.4 (3H,s), 2.9, 3.0 (total 3H, 2s), 3.6 (2H, s), 4.7, 5.2 (total 1H, 2q), 9.8(1H, s).

N-methyl-N-(3-methyldithio-propanoyl)-L-alanine (6 b). To a stirredsolution of 3-methyldithio-propanoic acid (4 b) (1.00 g, 6.57 mmol) indry THF (20 mL) at −10° C. under argon was added isobutylchloroformate(0.897 g, 6.57 mmol) and triethylamine (0.665 g, 6.57 mmol) and thereaction mixture was stirred for 15 minutes. A mixture ofN-methyl-L-alanine (0.677 g, 6.57 mmol) and triethylamine (1.33 g, 13.14mmol) in water (10 mL) was added and the reaction mixture was stirred atroom temperature overnight. The mixture was then diluted with water (50mL), acidified (aqueous HCl), and extracted with ethyl acetate (4×50mL). The combined organic layers were dried over sodium sulfate, thesolvent evaporated under reduced pressure and the residuechromatographed over silica gel eluting with methylene chloride/ethylacetate to yield 0.556 g (34%) white crystals: mp 98-100° C. ¹H NMR(CDCl₃) δ1.3 (3H, d), 2.2 (3H, s), 2.7 (4H, m), 4.5 (1H, q), 10.7 (1H,s). Anal. calculated for C₈H₁₅NO₃S₂:C, 40.49; H, 6.37; N, 5.90; mol wt.237.33. Found: C, 40.42; H, 6.41; N, 5.93.

N-methyl-N-(4-methyldithio-butanoyl)-L-alanine (6 c). To a stirredsolution of 4-methyldithio-butanoic acid (4 c) (0.200 g, 1.20 mmol) indry THF (10 mL) at −20° C. under argon was added isobutyl chloroformeta(0.164 g, 1.20 mmol) and triethylamine (0.121 g, 1.20 mmol) and themixture was stirred for twenty minutes. A mixture of N-methyl-L-alanine(0.124 g, 1.20 mmol) and triethylamine (0.243 g, 2.40 mmol) in water (5mL) was then added and the reaction mixture was stirred at roomtemperature for five hours. The reaction mixture was then treated asdescribed above for 6 b giving the title compound as white crystals(0.135 g, 44%): mp 92-93° C. ¹H NMR (CDCl₃) δ1.4 (3H, d), 2.0 (2H, m),2.3 (3H, s), 2.7 (4H, m), 2.9 (3H, s), 5.1 (1H, q), 10.5 (1H, s).

N-methyl-N-(5-methyldithio-pentanoyl)-L-alanine (6 d). To a stirredsolution of 5-methyldithio-pentanoic acid (4 d) (0.202 g, 1.12 mmol) indry THF (15 mL) at −40° C. under argon was added isobutyl chloroformate(0.153 g, 1.12 mmol) and triethylamine (0.113 g, 1.12 mmol) and thereaction mixture was stirred for 20 minutes at −10° C. A solution ofN-methyl-L-alanine (0.116 g, 1.12 mmol) and triethylamine (0.227 g, 2.24mmol) in water (5 mL) was then added and the mixture was stirred at 0°C. for five hours. The reaction mixture was treated as described abovefor 6 b affording the title compound as white crystals (0.196 g, 66%):mp 84° C. ¹H NMR (CDCl₃) δ1.4 (3H, d), 1.8 (4H, m), 2.4 (3H, s), 2.7(4H, m), 3.0 (3H, s), 5.2 (1H, q), 10.7 (1H, s).

N-Methyl-N-(3-phenyldithio-propanoyl)-L-alanine (6 e). A solution of3-phenyldithio-propanoic acid (5 a) (1.8 g, 8.4 mmol) in dry THF wasstirred vigorously under a nitrogen atmosphere and cooled to −15° C.Isobutyl chloroformate (1.2 mL, 9.25 mmol) and triethylamine (1.29 mL,9.25 mmol) were added and the reaction mixture was stirred at thistemperature for ten minutes. A solution of N-methyl-L-alanine (0.87 g,8.4 mmol) and triethylamine (1.29 mL, 9.25 mmol) in water (10 mL) wasthen added and the reaction mixture was stirred for fifteen minutes at−15° C. and then warmed to room temperature and stirred for anadditional period of 2.5 hours. 1 M HCl (10 mL) was added and thereaction mixture was extracted with ethyl acetate (4×50 mL). Thecombined organic layers were dried with Na₂SO₄, filtered, and evaporatedunder reduced pressure. The crude mixture was purified by columnchromatography on silica gel eluting with ethylacetate/hexane-containing 2% acetic acid to give 6 e a white solid (1.5g, 60%): mp 96-97° C. ¹H NMR (CDCl₃/TMS) δ1.4 (2H, d), 2.7-3.0 (7H, m),5.2 (1H, q), 7.2-7.6 (5H, m).

N-Methyl-N-[3-(4-nitrophenyldithio)-propanoyl]-L-alanine (6 f). To astirred solution of 3-(4-nitrophenyldithio)-propanoic acid (5 b) (0.100g, 0.386 mmol) in dry THF (10 mL) at −40° C. under argon was addedisobutyl chloroformate (0.053 g, 0.386 mmol) and triethylamine (0.039 g,0.38 mmol) and the reaction stirred at 0° C. for one hour. An aqueoussolution (5 mL) of N-methyl-L-alanine (0.040 g, 0.386 mmol) andtriethylamine (0.039 g, 0.386 mmol) was then added and the mixturestirred at 0° C. for five hours. The mixture was diluted with water (50mL), acidified (aqueous HCl), and extracted with ethyl ether (3×25 mL).The combined organic layers were dried (Na₂SO₄), and the solventevaporated under reduced pressure. The residue was chromatographed oversilica gel eluting with methylene chloride/ethyl acetate to yield 0.048g (36%) yellow crystals: mp 74-77° C. ¹H NMR (CDCl₃) δ1.4 (3H, d),2.6-3.4 (4H, m), 2.9 (3H, s), 5.1 (1H, q), 7.6-8.3 (4H, 2d).

N-Methyl-N-(3-methyldithio-butanoyl)-L-alanine (6 g). A solution of 4 e(3.52 g, 21.2 mmol) in tetrahydrofuran (20 mL) was cooled to −20° C.,and stirred under an argon atmosphere. Isobutylchloroformate (3.48 mL,21.2 mmol) and triethyl amine (3.7 mL, 25.4 mmol) were then added. Thereaction mixture was stirred at −20° C. for 15 min. A solution ofN-methyl-L-alanine (2.74 g, 26.6 mmol) in water (5 mL) and triethylamine(7.4 mL, 50.8 mmol) was added and the reaction warmed to roomtemperature and stirred for 2 h. The reaction mixture was diluted withwater (100 mL) and acidified to pH of 2 by addition of 1M HCl. Theproduct was extracted with ethyl acetate (5×50 mL). The combined organiclayer was dried with sodium sulfate, filtered and evaporated. Theresidue was purified by column chromatography over silica gel elutingwith ethyl acetate:hexanes (1:1, v/v) to give pure 6 g as a white solid(0.69 g, 12% yield). ¹H NMR (CDCl₃): δ1.3 (3H, d), 1.5 (3H, d), 2.41(3H, s), 2.8 (2H, m), 3.0 (3H, s), 3.5 (1H, m) and 5.3 (1H, m).

Methyldithio-N-methylcysteine (8 a). A solution of N-methylcysteine (7a) (Undhein, K., & Eidem, A., 23 Acta Chem. Scandinavica 3129-3133(1970)) (1.5 g, 11.1 mmol) in H₂O (20 mL) was stirred at roomtemperature, under an atmosphere of argon, and treated with a solutionof methyl methanethiol sulfonate (3.0 mL, 29.2 mmol) in ethanol (10 mL).The reaction mixture was stirred at this temperature for two hours andthen diluted with H₂O (100 mL) and washed with ether (4×40 mL). Theaqueous layer was acidified to pH 2 and passed through an Amberlite IRA93 (—OH form) column. The column was washed with water and the effluentwas collected and evaporated to dryness under reduced pressure to give awhite solid (1.2 g, 60%), mp 194-195° C. ¹H NMR (D₂O, TMS ext.standard): δ2.2(3H,s), 2.5(3H,s), 3.2(2H,d), 3.7(1H,q).

Methyldithio-N-methylhomocysteine (8 b). One of ordinary skill in theart would understand how to make methyldithio-N-methylhomocysteine (8 b)based on the method for making methyldithio-N-methylcysteine (8 a).

N-methylhomocysteine (7 b) can be made by the method previouslydescribed for N-methylcysteine (Undhein, K., & Eidem, A., 23 Acta Chem.Scandinavica 3129-3133 (1970)).

N-Acetyl-N-methyl-methyldithiocysteine (9 a). To a solution of glacialacetic acid (0.25 mL, 4.4 mmol) in dry THF (4 mL), at −20° C. under anatmosphere of N₂ were added, with stirring, isobutylchloroformate (0.57mL, 4.4 mmol) and triethylamine (0.61 mL, 4.4 mmol). The reactionmixture was stirred at this temperature for 20 minutes and then treatedwith a solution of methyldithio-N-methylcysteine (8 a) (0.4 g, 2.2 mmol)and triethylamine (0.45 mL, 3.3 mmol) in H₂O and then allowed to stirovernight. The mixture was then diluted with water (25 mL) and acidifiedto pH 2 with HCl and extracted with ethyl acetate (4×50 mL). Thecombined organic layer was dried with sodium sulfate and the solventevaporated under reduced pressure to give the product as a pale yellowsolid (0.2 g, 55%) mp 137-138° C. ¹H NMR (CDCl₃): δ2.1(3H,s), 2.3(3H,s),3.0(3H,s), 3.2(2H,d) and 4.8(1H,q).

N-acetyl-N-methyl-methyldithiohomocysteine (9 b). One of ordinary skillin the art will understand how to makeN-acetyl-N-methyl-methyldithiohomocysteine (8 b) based on the method formaking N-acetyl-N-methyl-methyldithiocysteine (9 a).

Step 1

Reduction of Ansamitocin P-3 (1 c) into Maytansinol

Ansanmitocin P-3 (1 c) was converted into maytansinol (2) by reductivehydrolysis. Ansamitocin P-3 (1 c) (3.2 g, 5.0 mmol) was dissolved inanhydrous THF (80 mL), and the solution was placed under an argonatmosphere, and cooled in a dry ice-acetone bath to −40° C.

To a separate flask was added a solution of lithium aluminum hydride (75mmol, 75 mL of a 1.0 M solution in THF). The solution was placed underan argon atmosphere and cooled to −40° C. A solution of anhydrousmethanol (9.1 mL, 225 mmol) in THF (40 mL) was added dropwise using adropping funnel. The temperature was maintained between −30 and −40° C.,and, after the addition was complete, the reaction mixture was stirredfor an additional period of 10 min. The resulting solution of lithiumtrimethoxyaluminum hydride (LiAlH(OMe)₃H) was transferred via a canulaover 10 min. into the chilled solution of ansamitocin P-3 (1 c). Thereaction temperature was maintained between −30 and −35° C. under argon,and stirred for 1 h.

The reaction was quenched by the addition of saturated sodium chloridesolution (100 mL), and extracted with ethyl acetate (4×400 mL). Thecombined ethyl acetate extracts were dried over sodium sulfate andfiltered. The solvent was evaporated under reduced pressure to givecrude maytansinol (2).

The crude product was dissolved in ethyl acetate and loaded onto asilica gel column packed in dichloromethane. The column was eluted witha step gradient starting with a mixture of dichloromethane:ethylacetate:methanol (77:23:0, v/v/v) and slowly increasing theconcentration of methanol from 0% to 10%. Fractions containing thedesired product were pooled and evaporated under reduced pressure togive pure maytansinol (71% yield) as a white solid.

The maytansinol was further purified by HPLC as follows. A Kromasil™cyano preparative HPLC column (250 mm×50 mm, 10 micron particle size)was equilibrated in a mixture ofhexanes:2-propanol:dichloromethane:ethyl acetate (72:3:18:7, v/v/v) at aflow rate of 150 mL/min. A solution of the maytansinol was injected ontothe column. Under these conditions, the maytansinol has a retention timeof 22 min and a purity of >98%.

Step 2

Esterification of Maytansinol (2) withN-methyl-N-methyldithioacetoyl-L-alanine (6 a)

A solution of N-methyl-N-methyldithioacetoyl-L-alanine (6 a) (4.2 mg,0.017 mmol) in dichloromethane (0.1 mL) was stirred under an argonatmosphere, and treated sequentially with a solution of maytansinol (2mg, 0.0035 mmol) in dichloromethane (0.2 mL), DCC (4.4 mg) indichloromethane (0.2 mL) and 1M zinc chloride (0.0035 mmol) in ether.The reaction was stirred for 75 min. The reaction mixture was thenfiltered and the solvent was evaporated. The residue was purified bypreparative TLC on silica gel, using 6% methanol in chloroform to givethe desired L-maytansinoid ester 10 a.

The compound 10 a can be more efficiently purified using a Diazem™ cyanoHPLC column (250 mm×10 mm, 10 micron particle size) equilibrated in amixture of hexanes:2-propanol:ethyl acetate (17:2:6, v/v/v) at a flowrate of 4.7 mL/min. ¹H NMR (CDCl₃) L-aminoacyl isomer (10 a): δ0.90(3H,s), 1.3 (6H, d,d) 1.46-1.52 (1H, m), 1.60 (3H, s), 2.30 (1H, d), 2.50(3H, s), 2.7 (1H, dd), 2.90 (1H, m) 3.15 (3H, s), 3.28 (3H, s), 3.3 (1H,d), 3.35 (3H, s), 3.50 (1H, m), 3.65 (2H, d), 3.77 (1H, d,), 4.02 (3H,s), 4.30 (1H, t), 4.82 (1H, dd), 5.15 (1H, q, J=7 Hz), 5.90 (1H, dd),6.25 (1H, s), 6.47 (1H, dd), 6.60 (1H, d), 6.75 (1H, d), 6.85 (1H, d).

Esterification of Maytansinol (2) withN-methyl-N-(3-methyldithio-propanoyl)-L-alanine (6 b)

A solution of N-methyl-N-(3-methyldithio-propanoyl)-L-alanine (6 b) (5.0g, 21.5 mmol) in dry methylene chloride (80 mL) was stirred under anargon atmosphere, and treated sequentially with solutions of DCC (4.58g, 22.2 mmol) in methylene chloride (54 mL), 1M ZnCl₂ in ether (4.4mmol), and maytansinol (2) (2.0 g, 3.5 mmol) in methylene chloride (80mL). The reaction mixture was stirred at room temperature for fourhours, and then filtered.

A Diazem™ cyano preparative HPLC column (250 mm×50 mm, 10 micronparticle size) was equilibrated in a mixture of hexanes:2-propanol:ethylacetate (17:2:6, v/v/v) at a flow rate of 150 mL/min. A solution of themixture of the L- and D-aminoacyl ester 10 of maytansinol (1 g in 25 mLethyl acetate) was injected onto the column. Under these conditions, thedesired L-isomer (10 b) has a retention time of 42 min, while theD-isomer elutes at 56 min. Optically pure product is obtained by thismethod, and the recovery is >90%.

¹H NMR (CDCl₃) L-aminoacyl isomer (10 b): δ0.84(3H, s), 1.11-1.23 (1H,m), 1.31 (3H, d, J=6 Hz), 1.35 (3H, d, J=7 Hz), 1.46-1.52 (1H, m), 1.68(3H, s), 1.97 (1H, d, J=9 Hz), 2.24 (1H, dd, J=12 Hz and 15 Hz), 2.30(3H, s), 2.65 (1H, dd, J=12 Hz and 15 Hz), 2.73-2.86 (2H, m), 2.90 (3H,s), 2.92-3.03 (2H, m), 3.08 (1H, d, J=9 Hz), 3.14 (1H, d, J=12 Hz), 3.28(3H, s), 3.39 (3H, s), 3.54 (1H, d, J=9 Hz), 3.72 (1H, d, J=13 Hz), 4.02(3H, s), 4.31 (1H, t, J=11 Hz), 4.82 (1H, dd, J=3 Hz and 12 Hz), 5.45(1H, q, J=7 Hz), 5.69 (1H, dd, J=9 Hz and 15 Hz), 6.25 (1H, s), 6.47(1H, dd, J=11 Hz and 15 Hz), 6.67 (1H, d, J=1.5 Hz), 6.77 (1H, d, J=11Hz), 6.85 (1H, d, J=1.5 Hz).

Esterification of Maytansinol (2) withN-methyl-N-(methyldithiobutanoyl)-L-alanine (6 c)

A solution of N-methyl-N-(methyldithiobutanoyl)-L-alanine (6 c) (8.9 mg,35.5 μmol) in dry methylene chloride (0.15 mL) was stirred under anatmosphere of argon, and treated sequentially with solutions of DCC (8.8mg, 42.6 μmol) in methylene chloride, 1M ZnCl₂ (7.1 μmol) in ether andmaytansinol (2) (4.0 mg, 7.1 μmol) in methylene chloride. The reactionmixture was stirred at room temperature for three hours, and thenfiltered and the filtrate was evaporated under reduced pressure,yielding a mixture of L- and D-aminoacyl esters of maytansinol. Thismaterial can be further purified using a Diazem™ cyano preparative HPLCcolumn (250 mm×50 mm, 10 micron particle size) equilibrated in a mixtureof hexanes:2-propanol:ethyl acetate (17:2:6, v/v/v) at a flow rate of150 mL/min as described above for 10 b.

Esterification of Maytansinol (2) withN-methyl-N-(phenyldithio-propanoyl)-L-alanine (6 e)

A solution of N-methyl-N-(phenyldithio-propanoyl)-L-alanine (6 e) (31.5mg, 105 μmol) in methylene chloride (0.4 mL) was stirred under argon andtreated sequentially with solutions of DCC (26 mg, 126 μmol) inmethylene chloride, 1M ZnCl₂ (17.7 μmol) in ether and maytansinol (2)(10 mg, 17.7 μmol) in methylene chloride (0.2 mL). The reaction mixturewas stirred at room temperature for three hours. The precipitate wasremoved by filtration and the filtrate concentrated under reducedpressure.

The mixture can be further purified using a Diazem™ cyano preparativeHPLC column (250 mm×50 mm, 10 micron particle size) was equilibrated ina mixture of hexanes:2-propanol:ethyl acetate (17:2:6, v/v/v) at a flowrate of 150 mL/min as described above for (10 b).

¹H NMR (CDCl₃) L-aminoacyl isomer (10 e): δ0.82 (3H, s), 1.11-1.25 (1H,m), 1.33 (3H, d, J=3 Hz), 1.61 (3H, s), 1.63 (3H, d, J=14 Hz), 2.19 (1H,dd, J=13 Hz and 15 Hz), 2.61 (1H, dd, J=12 Hz and 15 Hz), 2.78 (3H, s),2.68-3.03 (2H, m), 3.07 (1H, d, J=9 Hz), 3.20 (3H, s), 3.38 (3H, s),3.53 (1H, d, J=9 Hz), 3.63 (1H, d, J=13 Hz), 3.68 (3H, s), 4.01 (3H, s),4.30 (1H, t, J=11Hz), 4.79 (1H, dd, J=3 Hz and 8 Hz), 5.43 (1H, q, J=7Hz), 5.68 (1H, dd, J=9 Hz and 15 Hz), 6.23 (1H, s), 6.45 (1H, dd, J=12Hz and 15 Hz), 6.60 (1H, d, J=1.5 Hz), 6.75 (1H, d, J=12 Hz), 6.77 (1H,d, J=1.5 Hz), 7.22-7.40 (5H, m).

Esterification of Maytansinol (2) withN-methyl-N-(3-methyldithio-butanoyl)-L-alanine (6 g)

A solution of N-methyl-N-(3-methyldithiobutanoyl)-L-alanine (6 g) (23.2mg, 0.088 mmol) in dichloromethane (0.2 mL) was stirred under an argonatmosphere, and treated sequentially with a solution of maytansinol (5mg, 0.0088 mmol) in dichloromethane (0.2 mL), DCC (20.6 mg) indichloromethane (0.2 mL) and 1M zinc chloride (0.0088 mmol) in ether.The reaction was stirred overnight at room temperature. The reactionmixture was then filtered and the solvent was evaporated. The residuewas purified by preparative TLC on silica gel, using 6% methanol inchloroform to give the desired L-maytansinoid ester 10 g. The product 10g can be more efficiently purified using a_Diazem™ cyano preparativeHPLC column (250 mm×10 mm, 10 micron particle size) equilibrated in amixture of hexanes:2-propanol:ethyl acetate (17:2:6, v/v/v) at a flowrate of 4.70 mL/min as described above for (10 b).

Esterification of Maytansinol (2) with N-acetyl-N-methylmethyldithiocysteine (9 a)

A solution of N-Acetyl-N-methyl-methyldithiocysteine (9 a) (15.6 mg,0.07 mmol) in dry methylene chloride (0.45 mL) was stirred at roomtemperature under an argon atmosphere and treated sequentially withsolutions of 1M ZnCl₂ in ethyl ether (0.028 mmol), DCC (17.3 mg, 0.084mmol) in methylene chloride (0.2 mL), and maytansinol (2) (4.0 mg, 0.007mmol) in methylene chloride (0.1 mL). The reaction mixture was stirredfor three hours and then filtered and the filtrate evaporated underreduced pressure.

The residue can be further purified using a Diazem cyano preparativeHPLC column (250 mm×50 mm, 10 micron particle size) was equilibrated ina mixture of hexanes:2-propanol:ethyl acetate (17:2:6, v/v/v) at a flowrate of 150 mL/min as described above for (10 b).

Step 3

Reduction of Disulfide-Containing Maytansinoid

A solution of the disulfide-containing L-aminoacyl ester of maytansinol10 b (1.95 g, 2.5 mmol) in a mixture of ethyl acetate (140 mL) andmethanol (210 mL) was stirred at room temperature under an argonatmosphere, and treated with a solution of dithiothreitol (0.95 g, 6.2mmol) in 0.05 M potassium phosphate buffer (140 mL), pH 7.5, containing2 mM ethylenediaminetetraacetic acid (EDTA). The progress of thereaction was monitored by HPLC and was complete in three hours.

The completed reaction mixture was treated with a solution of 0.2 Mpotassium phosphate buffer (250 mL), pH 6.0, containing 2 mM EDTA, andthen extracted with ethyl acetate (3×600 mL). The organic layers werecombined, washed with brine (100 mL) and then dried over sodium sulfate.Evaporation of the solvent gave a residue of crude thiol-containingmaytansinoid 11 b.

The crude thiol-containing maytansinoid 11 b was purified by HPLC usinga preparative Diazem™ cyano HPLC column (250 mm×50 mm, 10 micronparticle size) that was equilibrated in a mixture ofhexanes:2-propanol:ethyl acetate (78.0:5.5:16.5, v/v/v) and ran at aflow rate of 150 mL/min. Thiol-containing maytansinoid 11 b eluted aspeak centered at 16 min. Fractions containing the product wereevaporated to give pure thiol-containing maytansinoid 11 b as a whitesolid (76% yield with a purity of 99%).

The presence of one mole of sulfhydryl group/mol product was confirmedusing Ellman's assay. The product was further characterized by NMRspectroscopy. ¹H NMR (CDCl₃): δ0.84 (3H, s), 1.33 (3H, d, J=5 Hz), 1.35(3H, d, J=5 Hz), 1.60 (3H, s), 1.68 (3H, s), 2.22 (1H, dd, J=3 Hz and 14Hz, 2.60-2.82 (2H, m), 2.88 (3H, s), 3.08-3.20 (2H, m), 3.25 (3H, s),3.39 (3H, s), 3.55 (1H, d, J=9 Hz), 3.71 (1H, d, J=12 Hz), 4.02 (3H, s),4.32 (1H, t, J=10 Hz), 4.81 (1H, dd, J=3 Hz and 12 Hz), 5.45 (1H, q, J=7Hz), 5.67 (1H, dd J=9 Hz and 15 Hz), 6.25 (1H, s), 6.47 (1H, dd, J=11 Hzand 15 Hz), 6.70 (1H, d, J=1.5 Hz), 6.75 (1H, d, J=11 Hz), 6.86 (1H, d,J=1.5 Hz).

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A process for preparing a thiol-containingmaytansinoid wherein the thiol group is part of the ester moiety at C-3,comprising the steps of: (1) conducting reductive hydrolysis of amaytansinoid C-3 ester with a reducing agent selected from the groupconsisting of lithium trimethoxyaluminum hydride (LiAl(OMe)₃H), lithiumtriethoxyaluminum hydride (LiAl(OEt)₃H) and lithium tripropoxyaluminumhydride (LiAl(OPr)₃H), to yield a maytansinol; (2) purifying themaytansinol to remove side products when present; (3) esterifying thepurified maytansinol with a carboxylic acid to yield a reaction mixtureof an L- and a D-aminoacyl ester of maytansinol; (4) separating theL-aminoacyl ester of maytansinol from the reaction mixture in (3); (5)reducing the L-aminoacyl ester of maytansinol to yield athiol-containing maytansinoid; and (6) purifying the thiol-containingmaytansinoid.
 2. The process of claim 1, wherein the reducing agent in(1) is lithium trimethoxyaluminum hydride.
 3. The process of claim 1,wherein the reducing agent in (1) is used in a concentration of fromabout 5 to 100 equivalents per mole of the maytansinoid C-3 ester. 4.The process of claim 1, wherein the reducing agent in (1) is used in aconcentration of from about 7.5 to 30 equivalents per mole of themaytansinoid C-3 ester.
 5. The process of claim 1, wherein the reducingagent in (1) is used in a concentration of from about 10 to 20equivalents per mole of the maytansinoid C-3 ester.
 6. The process ofclaim 1, wherein the reductive hydrolysis in (1) is conducted at atemperature of from about −80° C. to 0° C.
 7. The process of claim 1,wherein the reductive hydrolysis in (1) is conducted at a temperature offrom about −45° C. to −27.5° C.
 8. The process of claim 1, wherein thereductive hydrolysis in (1) is conducted at a temperature of from about−35° C. to −30° C.
 9. The process of claim 1, wherein the reducing agentin (1) is added over a period of from about 5 to 40 minutes.
 10. Theprocess of claim 1, wherein the reducing agent in (1) is added over aperiod of from about 7 to 20 minutes.
 11. The process of claim 1,wherein the reducing agent in (1) is added over a period of from about 8to 12 minutes.
 12. The process of claim 1, wherein the maytansinol ispurified in (2) by chromatography.
 13. The process of claim 12, whereinthe chromatography is silica gel column chromatography, preparativethin-layer chromatography on silica gel or cyano-bonded silica HPLCcolumn chromatography.
 14. The process of claim 12, wherein thechromatography is silica gel column chromatography.
 15. The process ofclaim 12, wherein the purification is performed at ambient temperature.16. The process of claim 12, wherein the maytansinol is purified to apurity of about 95%.
 17. The process of claim 1, wherein the carboxylicacid in (3) is selected from the group consisting ofN-methyl-N-methyldithioacetyl-L-alanine,N-methyl-N-(3-methyldithio-propanoyl)-L-alanine,N-methyl-N-(3-methyldithio-butanoyl)-L-alanine,N-methyl-N-(4-methyldithio-butanoyl)-L-alanine,N-methyl-N-(5-methyldithio-pentanoyl)-L-alanine,N-methyl-N-(3-phenyldithio-propanoyl)-L-alanine,N-methyl-N-(3-(4-nitrophenyldithio)propanoyl)L-alanine,N-acetyl-N-methyl-methyldithiocysteine andN-acetyl-N-methyl-methyldithiohomocysteine.
 18. The process of claim 1,wherein the carboxylic acid in (3) isN-methyl-N-(3-methyldithio-propanoyl)-L-alanine.
 19. The process ofclaim 1, wherein the esterification in (3) is conducted at ambienttemperature.
 20. The process of claim 1, wherein the esterification in(3) further comprises the use of dicyclohexylcarbodiimide and zincchloride.
 21. The process of claim 1, wherein the separating in (4) iscarried out by passing the reaction mixture over a cyano-bonded silicaHPLC column.
 22. The process of claim 1, wherein the separating in (4)is carried out at about 25° C.
 23. The process of claim 1, wherein thereduction in (5) uses dithiothreitol as the reducing agent.
 24. Theprocess of claim 1, wherein the reduction in (5) is carried out in amixture of ethyl acetate-methanol-aqueous buffer which is capable ofkeeping buffer salts, dithiothreitol, unreduced maytansinoids andreduced maytansinoids in solution.
 25. The process of claim 24, whereinthe mixture of ethyl acetate-methanol-aqueous buffer is 1:1.5:1, v/v/v,ethyl acetate:methanol:aqueous buffer.
 26. The process of claim 24,wherein the concentration of the thiol-containing maytansinoid is suchthat the thiol-containing maytansinoid remains soluble in ethylacetate-methanol-aqueous buffer.
 27. The process of claim 26, whereinthe concentration of the thiol-containing maytansinoid is about 4 g/L.28. The process of claim 1, wherein the reduction in (5) is carried outin an oxygen-free atmosphere.
 29. The process of claim 1, wherein thereduction in (5) is carried out at about 25° C.
 30. The process of claim1, wherein the purifying of the thiol-containing maytansinoid in (6) isby chromatography.
 31. The process of claim 30, wherein the purifying ofthe thiol-containing maytansinoid in (6) is by a cyano-bonded HPLCcolumn chromatography.
 32. The process of claim 31, wherein thechromatography is by a cyano-bonded HPLC column equilibrated and run inan organic solvent.
 33. The process of claim 32, wherein the organicsolvent is a mixture of hexanes:2-propanol:ethyl acetate.
 34. Theprocess of claim 33, wherein the organic solvent is a 78.0:5.5:16.5,v/v/v, mixture of hexanes:2-propanol:ethyl acetate.